The present study was designed to compare evaluation of skeletal muscle metabolism (vastus lateralis) evaluated by 31P-magnetic resonance spectroscopy (MRS) and biochemical analysis. During identical isometric knee extensor exercise protocols to fatigue in eight men, biopsy samples were taken at rest, peak exercise, and 32 s postexercise and 31P-MRS data were collected continuously for phosphocreatine (PCr), pH, ATP, and P(i) at 8- or 32-s intervals. There was no difference in ATP or pH measurements between the two techniques at rest, during peak exercise, or in recovery. Corresponding measurements of pH by the two techniques were closely related (r = 0.88, P < 0.01), and pH measured by 31P-MRS was closely related to muscle lactate accumulation (r = -0.84, P < 0.001). The level of PCr at peak exercise, expressed as a percentage of the baseline value, was not different between the two techniques (42 +/- 15 vs. 46 +/- 15%). The results indicate that, in skeletal muscle in normal subjects, 1) measurements of pH and PCr at rest and during exercise do not differ between the 31P-MRS and biopsy techniques and 2) muscle pH measured by 31P-MRS is closely related to lactate accumulation in men. Our data suggest that direct comparison of results of studies of exercise metabolism using these two techniques is warranted.